Composition and in vitro cytotoxicity of cellular infiltrates in rejecting human kidney allografts.

Xenogeneic immune RNA (I-RNA) extracted from the spleens and lymph nodes of guinea pigs previously immunized with a murine fibrosarcoma was able to convert normal mouse lymphocytes to effector cells specifically cytolytic to the same murine tumor in vitro. This effect of I-RNA was dose-dependent, and destroyed by treatment with RNase, but not with DNase or pronase. I-RNA was fractionated by ultracentrifugation on a 5–20% sucrose density gradient and the fraction capable of transferring cell-mediated cytotoxicity (CMC) was shown to have a sedimentation coefficient of 8–16 S. I-RNA was also fractionated by oligo(dT)-cellulose affinity chromatography and the active fraction was found to possess polyadenylic acid (poly(A)) sequences thus resembling messenger RNA. The immunological activity of the poly (A)-containing RNA fraction was tumor-specific and RNase-sensitive. In further experiments, I-RNA fractionated by sucrose density gradient ultracentrifugation was subsequently chromatographed. on an oligo(dT)-cellulose column. CMC was transferred only by the fraction which sedimented at 8–16 S and also contained poly (A).     

The STR252 – IVS10nt546 – VNTR7 phenylalanine hydroxylase minihaplotype in five Mediterranean samples

IVS10nt546 (IVS10nt-11g→a) is the most common molecular defect of the phenylalanine hydroxylase gene causing phenylketonuria in Mediterranean populations. Previous studies have proposed various and alternative hypotheses concerning the geographical origin and pattern of diffusion of this mutation in this area. In this study, this issue was re-examined on a large sample (149) of “Mediterranean” IVS10nt546 mutant alleles analysed with multiallelic intragenic polymorphisms. The analysis of intragenic microsatellite (STR) and minisatellite (VNTR) polymorphisms shows allelic heterogeneity of the IVS10nt546 mutation. Eight STR and three VNTR alleles were found in association with the splicing defect. Of the ten detected STR–VNTR combinations (“minihaplotypes”), we identified a predominant allelic association (VNTR7 – STR252) embedded in a RFLP-haplotype 6 background, which seems to correspond to the ancestral gene originating in the Turkey–Israel area. Analysis of both absolute and relative gene frequencies of the STR252 – IVS10nt546 – VNTR7 minihaplotypes, shows statistically significant (P < 0.02) variations and may suggest gene flow from Turkey and/or Israel to Italy and Spain. The associated migratory events need not be unique in time (and people) but seem to suggest they may be traced back to the expansion of the Neolithic culture and people, thus allowing dating of the origin of this mutation to at least 5000–10 000 years ago. Alternative hypotheses are discussed to explain, in light of the available historical and pre-historical evidence, the pattern of diffusion of the IVS10nt546 mutation in the Mediterranean basin. Received: 24 March 1997 / Accepted: 9 April 1997     

Drosophila Casein Kinase I Alpha Regulates Homolog Pairing and Genome Organization by Modulating Condensin II Subunit Cap-H2 Levels

The spatial organization of chromosomes within interphase nuclei is important for gene expression and epigenetic inheritance. Although the extent of physical interaction between chromosomes and their degree of compaction varies during development and between different cell-types, it is unclear how regulation of chromosome interactions and compaction relate to spatial organization of genomes. Drosophila is an excellent model system for studying chromosomal interactions including homolog pairing. Recent work has shown that condensin II governs both interphase chromosome compaction and homolog pairing and condensin II activity is controlled by the turnover of its regulatory subunit Cap-H2. Specifically, Cap-H2 is a target of the SCF^Slimb E3 ubiquitin-ligase which down-regulates Cap-H2 in order to maintain homologous chromosome pairing, chromosome length and proper nuclear organization. Here, we identify Casein Kinase I alpha (CK1α) as an additional negative-regulator of Cap-H2. CK1α-depletion stabilizes Cap-H2 protein and results in an accumulation of Cap-H2 on chromosomes. Similar to Slimb mutation, CK1α depletion in cultured cells, larval salivary gland, and nurse cells results in several condensin II-dependent phenotypes including dispersal of centromeres, interphase chromosome compaction, and chromosome unpairing. Moreover, CK1α loss-of-function mutations dominantly suppress condensin II mutant phenotypes in vivo. Thus, CK1α facilitates Cap-H2 destruction and modulates nuclear organization by attenuating chromatin localized Cap-H2 protein.     

Hybrid approaches to molecular simulation

Molecular dynamics (MD) simulation is an established method for studying the conformational changes that are important for protein function. Recent advances in hardware and software have allowed MD simulations over the same timescales as experiment, improving the agreement between theory and experiment to a large extent. However, running such simulations are costly, in terms of resources, storage, and trajectory analysis. There is still a place for techniques that involve short MD simulations. In order to overcome the sampling paucity of short time-scales, hybrid methods that include some form of MD simulation can exploit certain features of the system of interest, often combining experimental information in surprising ways. Here, we review some recent hybrid approaches to the simulation of proteins.     

Identification of novel Th2-associated genes in T memory responses to allergens

Atopic diseases are associated with hyperexpression of Th2 cytokines by allergen-specific T memory cells. However, clinical trials with recently developed Th2 inhibitors in atopics have proven disappointing, suggesting underlying complexities in atopy pathogenesis which are not satisfactorily explained via the classical Th1/Th2 paradigm. One likely possibility is that additional Th2-associated genes which are central to disease pathogenesis remain unidentified. The aim of the present study was to identify such novel Th2-associated genes in recall responses to the inhalant allergen house dust mite. In contrast to earlier human microarray studies in atopy which focused on mitogen-activated T cell lines and clones, we concentrated on PBMC-derived primary T cells stimulated under more physiological conditions of low dose allergen exposure. We screened initially for allergen-induced gene activation by microarray, and validated novel genes in independent panels of subjects by quantitative RT-PCR. Kinetic analysis of allergen responses in PBMC revealed an early wave of novel atopy-associated genes involved in signaling which were coexpressed with IL-4 and IL-4R, followed by a later wave of genes encoding the classical Th2 effector cytokines. We further demonstrate that these novel activation-associated Th2 genes up-regulate in response to another atopy-associated physiological stimulus bacterial superantigen, but remain quiescent in nonphysiological responses in primary T cells or cell lines driven by potent mitogens, which may account for their failure to be detected in earlier microarray studies.     

Cycle and duration of the seminiferous epithelium in puma (Puma concolor)

Puma or sussuarana (Puma concolor) is the second largest feline in the American continent and has an ample latitudinal distribution in very diverse habitats. In relation to its conservation status, the puma is considered an extinction-threatened species. The study of the testis morphology and the spermatogenic process in a species is fundamental for establishing the physiologic patterns that will make possible the selection of the protocols for assisted reproduction. A number of peculiarities associated with the reproductive biology of specific species such as the duration of spermatogenic process can be used to determine the frequency of sperm collection. Nine adult male pumas maintained in captivity were used to determine the relative frequency of stages in the seminiferous epithelium cycle. Three of them received intra-testicular injections of 0.1ml tritiated thymidine to determine the duration of the seminiferous epithelium cycle, and were subjected to biopsy 7 days later. The cycle of the seminiferous epithelium in puma was didactically described into eight stages by the tubular morphology method. The total duration of one seminiferous epithelium cycle in puma was calculated to be 9.89 days, and approximately 44.5 days are required for development of spermatozoon from spermatogonia. The duration of spermiogenesis, prophase and other events of meiosis were 14.08, 15.20 and 1.79 days, respectively. The relative frequency of the pre-meiotic, meiotic and post-meiotic phases were 3.98, 1.79 and 4.12 days, respectively.     

UDPglucose pyrophosphorylase from Xanthomonas spp. Characterization of the enzyme kinetics, structure and inactivation related to oligomeric dissociation

The genes encoding for UDPglucose pyrophosphorylase in two Xanthomonas spp. were cloned and overexpressed in Escherichia coli. After purification to electrophoretic homogeneity, the recombinant proteins were characterized, and both exhibited similar structural and kinetic properties. They were identified as dimeric proteins of molecular mass 60kDa, exhibiting relatively high specific activity ( approximately 80Units/mg) for UDPglucose synthesis. Both enzymes utilized UTP or TTP as substrate with similar affinity. The purified Xanthomonas enzyme was inactivated after dilution into the assay medium. Studies of crosslinking with the bifunctional lysyl reagent bisuberate suggest that inactivation occurs by enzyme dissociation to monomers. UTP effectively protects the enzyme against inactivation, from which a dissociation constant of 15microM was calculated for the interaction substrate-enzyme. The UTP binding to the enzyme would induce conformational changes in the protein, favoring the subunits interaction to form an active dimer. This view was reinforced by protein modeling of the Xanthomonas enzyme on the basis of the prokaryotic UDPglucose pyrophosphorylase crystallographic structure. The in silico approach pointed out two main critical regions in the enzyme involved in subunit-subunit interaction: the region surrounding the catalytic-substrate binding site and the C-term.     

唐鱼野生种群在海南岛的新发现及其生态资料

于2007年在海南岛发现了国家Ⅱ级重点保护动物唐(Tanichthys albonubes Lin) 群,属种均为海南新记录.该属全球只有两种,种与种群间都呈间断分布,对研究鲤科鱼类的系统发育及古地理等均有重要的科研价值.由于早年过度采集作观赏鱼,加上珠三角地区的急速都市化发展,唐鱼曾被认为是野外已灭绝种.近年被重新发现后,迄今只知分布于广东省珠三角地区零散地点和越南广宁省下龙湾附近.海南岛足这个珍稀种首次在亚洲大陆以外被发现的地点,亦是唐鱼野生种群已知纬度最低的分布点.发现海南唐鱼的地点是一条低地小河,水质清澈,水流缓慢,水生植物茂盛,与广东省报道的唐鱼生境类似.发现海南种群的小河鱼类丰富,全今记录有共生鱼类20种,包括大量掠食性物种,但唐鱼足该地的优势种之一.在不同月份都可以发现体长10mm以下的仔鱼,显示海南岛的唐鱼无明显的繁殖季节.海南岛跟亚洲大陆隔离历史长,其唐鱼与亚洲大陆种群在形态、分子水平上是否存在明显差异,不同种群问的系统发育关系如何,海南唐鱼的保护重要性是否更为突出,是目前正在研究的课题.     

A multi-layer method to study genome-scale positions of nucleosomes

The basic unit of eukaryotic chromatin is the nucleosome, consisting of about 150 bp of DNA wrapped around a protein core made of histone proteins. Nucleosomes position is modulated in vivo to regulate fundamental nuclear processes. To measure nucleosome positions on a genomic scale both theoretical and experimental approaches have been recently reported. We have developed a new method, Multi-Layer Model (MLM), for the analysis of nucleosome position data obtained with microarray-based approach. The MLM is a feature extraction method in which the input data is processed by a classifier to distinguish between several kinds of patterns. We applied our method to simulated-synthetic and experimental nucleosome position data and found that besides a high nucleosome recognition and a strong agreement with standard statistical methods, the MLM can identify distinct classes of nucleosomes, making it an important tool for the genome wide analysis of nucleosome position and function. In conclusion, the MLM allows a better representation of nucleosome position data and a significant reduction in computational time.     

A Preliminary Study of the Temporal and Spatial Biomass Patterns of Herbaceous Vegetation Consumed by Mountain Gorillas in an Afromontane Rain Forest

Although many animal species consume herbaceous vegetation found in African tropical forests, little is known of the temporal and spatial availability of these plants. From September 2004 to August 2005 we conducted a study that quantified the temporal and spatial biomass availability of 20 species of herbs frequently consumed by endangered mountain gorillas at two locations (Buhoma and Ruhija) in Bwindi Impenetrable National Park, Uganda. In general, the biomass of herbs varied over the study period, but these changes were relatively small. For 12 of 18 and nine of 11 species in Buhoma and Ruhija, respectively, herb biomass differed significantly among habitat types. Of the nine species found in both locations, seven species had a higher biomass at Ruhija, one species had a higher biomass at Buhoma, and one species showed no difference. These results demonstrate that herb biomass varied little temporally but spatial differences in herb biomass were more pronounced. Future studies should investigate the variables that may influence herb phenological patterns such as rainfall, light, soil quality, previous disturbance regimes, and animal foraging and trampling damage.